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ENZYMS OF MILK AND BUTTER 



BY 



R. W. THATCHER and A. C. DAHLBERG 



Reprinted from JOURNAL OF AGRICULTURAL RESEARCH 

Vol. XI, No. 9 : : : ; Washington, D, C, November 26, 1917 




PUBLISHED BY AUTHORITY OF THB SECRETARY OF AGRICULTURE, WITH THE COOPERATION 
OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS 



WASHINGTON s GOVERNMENT PRINTING OFFICE: 1917 






ENZYMS OF MILK AND BUTTER 



By R. W. Thatcher, Director, Minnesota Agricultural Experiment Station, and A. C. 
DahlbERG, Assistant Dairy Husbandman, Wisconsin Agricultural Experiment 
Station. 

INTRODUCTION 

The deterioration of butter during storage is often attributed to the 
enzyms in the buttermilk which it contains. There has been some 
experimental study of the matter; but most of the published results of 
these investigations yield little information concerning the actual enzym 
content of butter, because of the fact that the studies dealt chiefly with 
the chemical changes in the butter itself during storage, and sufficient 
care was not used to prevent possible contamination by microorganisms. 
In fact, a survey of the literature dealing with the enzyms of milk and 
its products shows a lamentable lack of clearness on the part of the 
investigators in distinguishing between the enzyms of milk itself and 
those which are due to bacterial infections. 

Rogers (16) 2 found lipase to be the cause of the increase in the acidity 
of canned butter on standing. Rahn, Brown, and Smith (15) stated 
that 

all (storage) butter investigated showed an increase of " amid nitrogen, " i.e., nitrogen 
not precipitated by copper sulphate, tannic acid, or phosphotungstic acid. 

Their methods were shown to be at fault by Rogers, Berg, Potteiger, 
and Davis (17), who could observe 

no evidence of an increase in soluble nitrogen in butter on long standing at o° F., 
even when the conditions of manufacture were most favorable to such changes. 

They could detect no proteolysis in buttermilk held a long period of 
time in cold storage to which 18 per cent of sodium chlorid had been 
added, but active bacterial proteases, pepsin, and trypsin were not 
completely inhibited in their action by these adverse conditions. Lac- 
tose, they concluded, was oxidized only when a trace of iron and peroxid 
were added. 

During the summer of 191 5 one of us (Dahlberg), as a part of his 
duties in the Division of Dairy and Animal Husbandry of the Minnesota 
Agricultural Experiment Station, prepared several lots of butter under 
carefully controlled conditions of manufacture and placed them in stor- 

1 Published, with the permission of the Director, as Paper No. 71 of the Journal Series of the Minne- 
sota Agricultural Experiment Station. 

2 Reference is made by number (italic) to " Literature cited," pp. 448-450. 

Journal of Agricultural Research, Vol. XI, No. 9 

Washington, D. C. Nov. 26, 191 7 

kt Key No. Minn.— 23 

(437) 



43 & Journal of Agricultural Research vol. xi, no. 9 

age, in order to study the effect of varying methods of manufacture and 
storage upon the keeping qualities of the butter. From time to time 
studies of the bacterial development in the stored butter were made. 
The results of this work have recently been published elsewhere (25). 

Since the conditions of manufacture and the bacterial development 
in these butters were known, they afford excellent material for a study 
of the enzym content of the butter after storage, and such a study was 
accordingly undertaken. 

REVIEW OF PREVIOUS WORK 

The following types of enzyms have been reported to be normally 
present in cow's milk: an amylase (27), a lipase (9), proteases {1-5, 7, 22) 
a peroxidase (ig) y salolase (22), catalase (12, 18, 20), reductase (20), 
and a lactose-fermenting enzym (21). Vandevelde (22), however, 
concludes that no lipase can be found in fresh milk and that salolase is 
present in only very slight amounts; while Grimmer (10), as a result of 
his study of the enzyms of the mammary glands, concludes that salolase, 
peroxidase, and catalase are the only enzyms which are normally secreted 
with milk. Further, Van de Velde and Landsheere {23) found no 
amylase in milk and criticize Spolverini's observations concerning 
peroxidase in milk, holding that its presence there is due to bacterial 
contamination. 

These reports indicate something of the confusion which exists 
with reference to the normal enzyms of milk. The contradictory evi- 
dence which has been presented undoubtedly results, in part at least, 
from the rather crude methods of study of enzym action which were in 
use at the time when these investigations were in progress. Unfortu- 
nately, little attention has been given to these matters during recent 
years, when more refined methods of study might have given more 
concordant results. 

The question as to whether any enzyms which may be present in 
milk will be carried down with the butter in churning has not been 
generally discussed. Kooper (13) states that during butter making 
catalase remains behind in the buttermilk and is therefore not a direct 
constituent of butter fat. It might be assumed, however, that any 
buttermilk which remains in the butter would contain the same enzyms 
that were present in the original mil^k. In fact, Hesse (12), who found 
small amounts of catalase in butter, calculated his results on the basis 
of its buttermilk content. Furthermore, since butter fat is undoubtedly 
an emulsion colloid, it might be assumed to carry with it into the butter, 
in the form of absorption complexes, large proportions of the enzyms 
originally present in the milk. But no definite information concerning 
these matters, other than that cited in the opening paragraphs of this 
paper, has been published. 



xov. 26, 191- Enzyms of Milk and Butter 439 

EXPERIMENTAL WORK 
I. INVESTIGATIONS CONCERNING GALACTASE 

The normal proteolytic enzym of milk, which aids in the slow decom- 
position of milk proteins into peptones, amino acids, and ammonia, was 
given the name "galactase" by Babcock and Russell (1-3). This name 
is unfortunately not in accord with the present system of nomenclature, 
and the enzym ought properly to be called "casease" or "lactalbumi- 
nase"; but, since the name suggested by Babcock and Russell has come 
into common use and its significance is properly understood, we decided 
to continue its use. 

Before undertaking the proposed studies of the galactase content of 
milk, cream, skim milk, buttermilk, and butter it was necessary to 
determine upon certain details of technic which had not been satisfac- 
torily worked out by former investigators. This preliminary work may 
be briefly summarized as follows: 

(a) Length of period of action. — Previous experimental data 
give quantitative measures of proteolytic activity for periods varying 
from 1 hour for certain pepsin studies (8) up to the practically prohibitive 
time of nearly 3 years for galactase (14) . The time in which most investi- 
gators have allowed galactase to act, in order to obtain comparative 
results, has varied from 30 days to 1 year. This appears to be unneces- 
sarily long. The analytical data presented by Babcock and Russell give 
an average increase in the soluble-nitrogen content of a number of sam- 
ples of skim milk, when expressed as percentage of the total nitrogen, of 
13.49 P er cent £° r tne fi rst 8 days and only 5.66 per cent for the following 
12 days. One sample of skim milk increased 26 per cent in soluble nitro- 
gen during the first 7 days, 2 per cent the following 7 days, and to increase 
the soluble nitrogen content another 26 per cent required a period of 3 
days. There is very little difference in the comparative galactase con- 
tent of various skim milks when analyses made at the end of 7 days or 
of 6 months are compared. In the present work the period of action 
was limited to 4 days, since it was found that the soluble nitrogen in 
skim milk increased 10 or 12 per cent during this time. 

(6) Measurement of hydrolysis by means of the ninhydrin 
reaction. — The ninhydrin method, as proposed by Harding and 
MacLean (11) for the determination of amino-acid or nitrogen, was com- 
pared with the official method of the Association of Official Agricultural 
Chemists (26) for determining the increase in soluble nitrogen in differ- 
ent samples of milk due to protease action (Table I). In the ninhydrin 
determination the sample was diluted 1 to 10, and 1 c. c. of this used in 
each analysis, but the results were calculated to the 10 c. c. basis. Bac- 
terial action w T as inhibited by 0.5 per cent of chloroform. All samples in 
all the work reported in this paper were incubated at 40 C. for four days. 



440 



Journal of Agricultural Research 



Vol. XI, No. 9 



Table I. — Results with ninhydrin method of estimating 

teins of milk 



rate of hydrolysis of the pro- 



Sample. 


Total 
nitro- 
gen. 


Initial 
soluble 
nitro- 
gen. 


Initial 
ninhy- 
drin 
nitro- 
gen. 


48-hour 
soluble 
nitro- 
gen. 


48-hour 
ninhy- 
drin 
nitro- 
gen. 


Increase 

in 
soluble 
nitro- 
gen. 


Increase 
in nin- 
hydrin 
nitro- 
gen. 


Boiled skim milk 


Per cent. 
O. 0586 

.0586 
.0586 


Per cent. 
O. OO33 

. 0108 
. 0108 


Per cent. 
O. 0029 

. OO29 
. 0029 


Per cent. 
0.0039 

.0137 
.0177 


•Per cent. 
O. OO32 

.0031 
. 0032 


Per cent. 
I. 02 

4-95 
11.77 


Per cent. 
0.51 

■34 
•5i 


Skim milk + 3 per cent of 
sodium chlorid 


Skim milk 





The ninhydrin method indicated no increase in nitrogen when there 
was an actual increase of 11.5 per cent in the soluble nitrogen. This 
was further substantiated by work on a pure casein solution. This gave 
only a trace of nitrogen by the ninhydrin method, with no increase after 
evident proteolysis had taken place. This method of estimating the 
rate of hydrolysis of proteins was therefore abandoned as not being 
applicable to investigations with milk proteins. 

(c) Th# influence of chloroform on proteolysis. — To prevent 
bacterial action in the samples, chloroform was added in amounts found 
ample by Harding and Maclean (jj) and Van Slyke (24). Skim milk 
to be analyzed at varying intervals extending over a long period of time 
was treated with 1 per cent chloroform, which, according to the above 
investigators and Babcock and Russell (5), should have only slightly 
inhibited proteolysis. As shown in Table II, the soluble nitrogen of 
this skim milk did not increase on standing. In verifying this result 
the soluble nitrogen was obtained by a Kjeldahl determination of the 
nitrogen in the total filtrate from the casein precipitation. Closer 
agreement in the duplicates could be obtained from the filtrates than 
from the casein itself. 



TablB II. — Influence of chloroform on the proteolysis of milk as indicated by soluble 

nitrogen 



Sample. 


Chloro- 
form. 


Total 
nitrogen. 


Initial 

soluble 

nitrogen. 


Soluble 

nitrogen 

after 4 

days. 


Increase 
in soluble 
nitrogen as 
per cent 
of total. 


i . Skim milk 


Per cent. 
\ 1.0 

\ 1. 

I. 

( - 5 

1 1. 

I. 


Per cent. 
O. 0583 
•0583 
.0531 
•Q53 1 
. 0406 
. 0406 
•0547 
.0551 

.0463 


Per cent. 
O. 0129 
.0130 
.0115 
.0115 
. 0119 
. OI 19 
. 0106 

• OI2 5 
.0125 
. 0064 


Per cent. 
O. 0183 
. 0114 
. 0170 
. 0I20 
.0136 
.0103 
. 0079 
. 020I 
.0174 
.0059 


9. 26 


2 . Skim milk 


-2. 74 

io-35 


3. Skim milk 


.94 
4. 18 


4. Skim milk 


-3-94 

-4-93 

13.60 


5. Buttermilk, neutral 


6. Buttermilk, neutral 


9. 80 
— 1.08 







Nov. :6, 1917 



Enzyms of Milk and Butter 



44i 



Sample 5 was buttermilk obtained from a small hand churning and 
probably contained 1 per cent of butter fat. Since fat destroys the 
anesthetic property of chloroform by combining with it, the results of 
No. 5 should not be considered. In every other case 1 per cent of chlo- 
roform in skim milk or buttermilk completely inhibited proteolysis. 

(d) The influence of sodium chIvORId on proteolysis. — The pos- 
sibility that the sodium chlorid present in ordinary butter might inhibit 
proteolytic activity led us to make a study of the effect of varying per- 
centages of sodium chlorid added to skim milk upon the rate of produc- 
tion of soluble nitrogen by galactase. After the period of incubation 
the unchanged casein was precipitated from the milk by means of dilute 
acetic acid and the soluble nitrogen in the filtrate determined by the 
Kjeldahl method. The samples were preserved during the incubation 
by 0.75 per cent chloroform. The following results were obtained 
(Table III). 



Table III. — Influence of sodium chlorid on the proteolysis of milk (ivit 

chloroform) 



°-75 P er cen t °f 



Sodium chlorid added. 



Total 
nitrogen. 



Initial 
soluble 
nitrogen. 



Soluble 
nitrogen 

after 
21 days. 



Soluble 
nitrogen 

after 
72 days. 



Increase. 



Per cent 

0.0 boiled 

.0 

•5 

1.0 

*5-° 

20.0. 



Per cent. 
O. 0561 
.0561 
.0561 
• 56l 
•0534 
.0528 



Per cent. 
O. 0055 
. OI2I 
. OI2I 
. OI2I 
. OI2I 
. OI2I 



Per cent. 
o. 0051 



OI42 
OI20 



Per cent. 
o. 0034 
. 0198 
. 0171 
.0131 
. OIIO 
.OIO3 



Per cent. 

~3- 74 

13- 7 2 

8.91 

1.78 

— 2. 06 

-3.40 



The effect of sodium chlorid is marked; both 15 and 20 per cent, the 
concentration of the salt brine in butter, stopped all proteolysis. The 
very slight increase in the soluble nitrogen content of the skim milk 
containing 1 per cent of the salt is partiallv due to the excessive chloro- 
form used, as the following tests, in which only 0.5 per cent of the anes- 
thetic was used, will show (Table IV). 



Table IV.- — Influence of sodium chlorid on the proteolysis of milk {with 0.5 per cent of 

chloroform) 



Sodium chlorid added. 


Total 
nitrogen. 


Initial 

soluble 

nitrogen. 


Soluble 
nitrogen 

after 
S days. 


Increase. 


Per cent. 
0.0 boiled 


Per cent. 

O. 0586 

.0586 

.0586 

.0586 


Per cent. 

O. OO33 

. 0108 

. 0108 

. 0108 


Per cent. 

O. 0034 

. 0242 

. 020I 

• OI43 


Per cent. 
O. 51 


.0 


22.86 


.8 


I5-87 
5-97 


7.0 





442 



Journal of Agricultural Research 



Vol. XI, No. 9 



The actual checking of the increase in soluble nitrogen due to the 
addition of i per cent of sodium chlorid was approximately 10 per cent 
in either case, but 3 per cent of the salt did not entirely prevent proteo- 
lysis. 

(e) Occurrence of galactase in mii,k during process of butter 
making. — In following the protease of the fresh milk to the finished 
butter, the reaction of the various products was in every case brought 
to that of the fresh milk — that is, 10 c. c. were exactly neutralized by 
1.5 c. c. of N/10 alkali when phenolphthalein was used as an indicator, 
by dipping a stick of sodium hydroxid into the sample. If the sample 
became too alkaline, it was neutralized by some of the original sample 
so that dilution was avoided. The various samples were treated with 
chloroform in the following proportions: The skim milk, buttermilk, and 
bowl contents, 0.5 per cent; the milk, 2.5 per cent; and the cream, 
which contained 23 per cent of butter fat, 5 per cent. The "bowl con- 
tents" was an emulsion of the slime in the wash water held in the bowl. 
An equal volume of boiled skim milk was added as a substrate, but the 
increase in the soluble nitrogen was calculated on the basis of the bowl 
contents alone. The "cream during ripening" refers to the proteolysis 
occurring during the 10 hours it was ripened at 85 ° F. and the 20 hours 
it was held at 54 previous to churning. 

Table V. — Presence and concentration of galactose in milk and butter as indicated by the 

increase of soluble nitrogen 



Sample. 



Total 
nitrogen. 



Initial 

soluble 

nitrogen. 



Soluble 
nitrogen 

after 
4 days. 



Increase. 



Initial 

percentage 

of total 

nitrogen 

as casein. 



Skim milk 

Whole milk 

Cream 

Bowl contents 

Cream during ripening 
Cream after ripening. . 
Buttermilk 



Per cent. 
o. 0583 

0544 
O403 
O189 
O403 
O403 
055I 



Per cent. 
O. 0129 
.0113 
. 0076 
. OO46 
. 0076 
. 0081 
.0125 



Per cent. 
O. 0183 
. 0198 
. 0142 
. O202 
. 0081 
.0139 
. 020I 



Per cent. 
II. 10 
15.62 

l6 -37 

114. 28 

1.66 

14-39 
13.60 



77.87 

79- 2 3 
81. 14 

89-93 



It appears from these data that in separating milk, galactase is taken 
out of the skim milk part, slightly increased in the cream, and highly 
concentrated in the separator slime. While no relationship exists be- 
tween the increase in soluble nitrogen and the total nitrogen, it is evident 
that the factors at work during milk separation which increase the per- 
centage of casein in the total nitrogen also increase the galactase con- 
tent. The cream underwent a slight proteolytic digestion during the 
ripening process, but the proteolysis after souring and neutralization 
was less than that of the sweet cream. This indicates * that the chief 



xov. 26,1917 Enzyms of Milk and Butter 443 

proteolytic enzym of milk is not of bacterial origin, as Olson (14) recently 
reported. 

(/) Amount of galactase contained in butter. — The butter 
used in the experiments represented both good and bad qualities. The 
"fresh dairy" butter was made from the cream obtained from the milk 
of the University Farm herd, and was soured spontaneously without 
pasteurization. The "fresh creamery" butter was made in a cooper- 
ative creamery from sweet pasteurized cream ripened by a commercial 
starter. Both of these butters were of extra-fine quality. The "stored 
dairy" butter was the same as the "fresh dairy," except that it had 
been held in cold storage for eight months. The "fresh centralized" 
butter was made from the sour cream just as it was received by a central 
creamery. The last two butters mentioned were of poor quality. The 
"stored centralized" butter was made in the same way as the "fresh 
centralized," but had been held eight months in cold storage. It was of 
extremely poor quality. 

No effort was made to obtain a pure enzym extract from the butter. 
A known weight, usually 400 gm., of butter was melted at 45 ° C. in two 
long glass tubes about an inch in diameter. After the separation was 
complete the clear fat was hardened by immersing the tubes in cold 
water, and the curd solution was then washed out. If an excess of fat 
remained in the extract, it was removed by rewarming and centrifuging. 
This extract was then dialyzed at a temperature never exceeding i3°C, 
in a parchment dialyzer, until free of sodium chlorid, the last six or eight 
hours of dialysis being with distilled water. This curd was made up to a 
given volume and used at once in the various tests. 

The acetone-ether method of obtaining a fat-free, dry powder was 
also tried, but so high a percentage of sodium chlorid was left in' the 
powder that dialysis was necessary. Consequently this method had no 
advantages over the other, and the enzyms probably would have been 
weakened by the precipitation. 

The casein was precipitated by the optional official method (26, 
p. 118), rather than the official method because filtering was often more 
rapid, the volume to be filtered much less, and a clear filtrate more easily 
obtained. (Thirteen analyses of chloroformed-skim milk and butter- 
curd extracts gave an average of 0.00104 gm. more nitrogen in the form 
of casein by the use of alum as the precipitant; hence, the methods should 
not be used interchangeably.) A clear filtrate was more easily obtained 
and an excess of substrate assured by the addition of boiled skim milk 
to the curd solutions. In a few cases the filtering had to be done on a 
"Biichner funnel" through three filter papers and repeated 10 to 20 
times to obtain a perfectly clear filtrate. Bacterial action was prevented 
by 1 per cent chloroform instead of 0.5 per cent because the extracts 
contained considerable fat (Table VI). 



444 



Journal of Agricultural Research 



Vol. XI, No. 9 



Table) VI. — Presence of galactase in butter, as indicated by the increase in soluble 

nitrogen 



Kind of butte 



Concentration. 


Initial 


Soluble 






Ratio, 


Ratio, 


curd ex- 


soluble 


after 4 
days. 


Increase. 


butter 


tract to 


nitrogen. 




to curd 


boiled 






extract. 


skim milk 
added. 












Gm. 


Gm. 


Gm. 


2 : i 


50:100 


O. 0098 


O. 0108 


0. 0010 


2 : i 


50: 100 


. OOQI 


. 0091 


. OOOO 


2:1 


50: lOO 


. 0080 


. OO90 


. OOOO 


2: 1 


50:100 


. 0070 


.0045 


.0025 


3 ; 2 


5 o: 5° 


.0043 


.0063 


. 0020 


2:1 


50:50 


. OO42 


. 0078 


.0036 


2:1 


50:50 


.0039 


.0036 


.0003 


3 ; 2 


5o : 5o 


. 0049 


.0044 


.0005 



Increase 
for 10 gm. 
of butter. 



Fresh dairy 

Fresh dairy, boiled 

Fresh dairy 

Fresh dairy, boiled 

Stored dairy 

Stored centralized 

Stored centralized boiled 
Fresh creamery 



Gm. 

o. 0015 

. OOOO 

.0015 

. OOOO 

. 0027 

.0036 

. OOOO 

. OOOO 



The two samples of fresh dairy butter gave uniform and measurable 
proteolytic action, while one sample of fresh pasteurized creamery butter 
showed no digestion of the casein. The increase in the soluble nitrogen 
of the stored butter was nearly twice that of the fresh butter. 

11. lipase: content of butter 

Lipase hydrolyzes fats into alcohols and fatty acids, causing an in- 
crease in the acidity of the media acted upon. The increase in the 
acidity of butter on standing is supposed to be due to this enzym (16). 

The curd solutions used in testing for lipase activity were just half 
the volume of the butter. The substrate was either 5 per cent of butter 
fat or olive oil, and the preservative was 2.5 per cent of chloroform. 
Ten c. c. aliquots of the extracts in 10 c. c. of neutral 95 per cent alcohol 
were titrated against N/40 sodium hydroxid, with phenolphthalein as 
indicator. 

Table VII. — Presence of lipase in butter as shown by the increase of acidity 



Kind of butter. 



Initial 
acidity. 



Acidity 
after 4 days 



Increase 
overboiled. 



Fresh dairy, boiled 

Fresh dairy 

Fresh unsalted dairy, boiled 

Fresh unsalted dairy 

Stored unsalted dairy 

Fresh centralized 

Stored centralized 



6. Fresh creamery 



>er cent. 
O. 2 



Per cent. 

i-3 

1. 1 

5-3 
6.6 

5-5 
3-o 

3-5 

2. 2 



Per cent. 

0. O 
. O 

. o 

• -3 
. o 

•3 

1. i 

•4 



Nov. 26, 1917 



Enzyms of Milk and Butter 



445 



The extracts from the unsalted butters were not dialyzed. This 
accounts for their higher initial acidity. 

Lipase activity during four days at 40 C. was too small to be accurately 
measured, except in the extract from the stored centralized butter. 

III. OXIDASE CONTENT OF MILK AND BUTTER 

Since the investigations of oxidase activity in milk have been limited 
to a few easily oxidized substances and since oxidases show a specificity 
toward chemicals in their action, it was thought desirable to investigate 
by Bunzel's method the action of milk on several of the common chro- 
mogens (7). Four c. c. of milk were used in each test. Negative results 
were obtained with paraphenylene diamine, phenolphthalein, hydro- 
quinon, pyrocatechin, phloroglucin, a-naphthol, and para-, meta-, and 
ortho-cresols. Positive results were obtained with metol and pyrogallol ; 
but since the action of the latter was about 20 per cent the greater, 
pyrogallol alone was used for the final determinations. The results given 
in Table VIII were obtained in duplicate, the duplicates agreeing very 
closelv. 



Table VIII. — Estimation of oxidase in milk as shown by the oxygen absorption of 

pyrogallol 

[Readings are expressed as millimeters of mercury, each millimeter representing an absorption of 0.025 c. c. 

of oxygen.] 





Time. 


Skim milk 

1. 


Whole milk. 


Skim milk 2. 




Raw. 


Boiled. 


Raw. 


Boiled. 


60 


Minutes. 




O. 16 
.40 
•56 
.70 

•85 
•97 

1.70 
1.97 
2. 25 

2.32 


0. OO 

. OO 
. OO 

.16 

. 20 

.27 
.40 
.91 

1. 22 
I.48 

1 -55 


O. 00 

. CO 

•17 
.17 
•35 
.40 


O. OO 


80 






IOO 






120 


0-95 
I.07 

I.28 
i-33 








160 




180 




•200 






360 








420 








460 



















There was no agreement in the results obtained from the skim milk 
from the same herd on different days ; boiling failed to completely inhibit 
the activity of the whole milk, and the action was very slow in starting. 
It can not be caused by a normal milk oxidase. In every case a browning 
of the samples was quite marked before oxygen absorption started. 

Seven curd solutions from different butters gave no action with 
pyrogallol. 



446 



Journal of Agricultural Research 



Vol. XI, No. 9 



IV. CATALASE CONTENT OF MILK AND BUTTER 

Storch's method of measuring the catalase of milk, as given by Barthel 
(6) , gave very closely agreeing duplicate results and was used in this work 
(Table IX). In milk the liberation of oxygen ceased in four hours. The 
curd extracts were equal in volume to the original butter. Five per 
cent of commercial hydrogen peroxid was added to the samples to be 
tested, and the concentration of peroxid in the boiled samples at the 
end of four hours were used as the basis for calculation. 

Table IX. — Catalase content of milk and butter as shown by reaction with nydrogen 

Peroxid 



Sample. 



i. Boiled milk 

Milk 

2. Boiled fresh dairy butter 

Fresh dairy butter 

3. Stored dairy butter 

4. Fresh centralized butter 

5. Stored centralized butter 

6. Fresh pasteurized creamery butter 



Hydrogen peroxid con- 
tent after 4 hours. 



Boiled. Unboiled. 



Gm. 
O. 0054 



0054 



Gm. 



O. 0039 



0049 
0049 
0026 
0039 
0048 



Reduction 
of hydro- 
gen peroxid 
in unboiled 
milk or 
butter. 



Gm. 



0005 
0005 
0028 
0015 
0006 



The fresh centralized butter did not contain as much catalase as the 
results show, because a clear filtrate was not obtained.. Every sample 
of butter contained catalase. That the centralized butter gave higher 
results than the other butter and as high as milk itself agrees with the 
belief that there is some relationship existing between catalase activity 
in milk and bacterial growth. Storage did not diminish its activity. 

V. PEROXIDASE CONTENT OF MILK AND BUTTER 

The common Storch test for heated milk was used on the butter 
extracts with the following results : 

Table X. — Color changes shoiving the peroxidase content of milk and butter 







Time of heating (ir 


minutes). 









5 


10 


20 


30 


1. Fresh dairy milk 
boiled. 
Fresh dairy milk 


White... 
. ..do. .. . 


White... 

Gray . . . 
White... 
Gray . . . 
White... 
...do.... 


White 

Gray 

White 

Gray 

Faint gray . 
White 


White 

Gray 

Faint gray . 

Gray 

Faint gray . 
... do 


White. 

Gray. 

Faint gray. 
Gray. 
Faint gray. 
Do. 


2 . Stored dairy milk 


...do.... 


3 . Fresh centralized .... 


. ..do.... 


4. Stored centralized. . . . 


. . .do. .. . 


5. Fresh pasteurized 
creamery. 


. ..do... . 













xov. 26, 1917 Enzyms of Milk and Butter 447 

All the samples gave some color change, but for the storage butters 
and the pasteurized creamery butter it was very slight. Milk diluted 
1 to 160 with distilled water and to which 16 per cent of boiled milk was 
added gave a gray color similar to that of the fresh dairy butter; hence 
the peroxidase content of butter is very small. 

COMPARISON OF MILK AND BUTTER ENZYMS ON THE BASIS OF TQTAL 

NITROGEN 

The fat in butter acts as so great a diluent for the water-soluble con- 
stituents that a direct comparison of the enzyms of milk and butter 
is misleading. The more logical comparison is on the basis of the total 
nitrogen, since the proteins and enzyms exist in the same colloidal 
state and ought to be carried into the butter in the same proportions 
as they exist in the cream. The total protein of the fresh dairy butter 
was 0.55 per cent, that of the milk from which it was made 3.47 per 
cent, so the enzymic activities in the butter were multiplied by 6.31. 
On taking the milk as the standard and its enzymic content as 1 , butter 
was found to contain the following amounts of the various enzyms 
which were studied : 

Galactase 1. 100 j Catalase 2. 000 

Oxidase o. 000 | Peroxidase o. 008 

The galactase content of the butter was approximately equal to that 
of the milk, the catalase content was double, but the peroxidase content 
was very much less, and no oxidase activity could be detected. 

RELATION OF ENZYMS IN BUTTER TO DETERIORATION DURING 

STORAGE 

As mentioned in the introductory paragraphs of this paper, dete- 
rioration in quality of butter during storage has been considered by 
some investigators to be due to the action of enzyms contained in it. 
Fat-splitting (lipase) or protein-hydrolyzing ("galactase" or casease) 
enzyms have been suggested as possible agents in causing deterioration. 
Our work leads us to conclude that lipases are present in butter in very 
small amounts, if at all, and that they could not be conceived to be 
sufficiently active at the low temperature used in butter storage to 
cause any appreciable change in the quality of the butter. The protein- 
hydrolyzing enzym we found to be completely inhibited by sodium 
chlorid in the concentrations which are present in the water contained 
in all normally salted butters. This fact, together with the known 
inhibiting effect of low temperatures upon proteolysis by enzyms makes 
it impossible that the hydrolysis of proteins in the butter by enzyms 
plays any part in deterioration changes. We conclude, therefore, that 
enzyms are not to be considered as a factor in the deterioration of butter 
in cold storage. 



448 Journal of Agricultural Research vol. xi. no. 9 

SUMMARY 

(1) Proteolysis in skim milk was completely inhibited by 1 per cent 
of chloroform and by 15 per cent of sodium chlorid. Galactase can not 
act in normal butter because of the high salt content. 

(2) In the separation of milk the factors which increase the percentage 
of casein in the total nitrogen also increased the galactase content. 
The ripening of cream did not increase the rate of proteolysis. 

(3) No oxidase was found in milk or butter. 

(4) Only one sample of butter gave any evidence of lipase at the 
end of four days at 40 C. 

(5) The enzym content of butter is very small, because of the high 
dilution in fat. Expressed on the basis of total nitrogen the butter 
examined contained as much galactase as fresh whole milk, twice as 
much catalase, but only one one hundred and sixtieth as much perox- 
idase. 

(6) The cold storage of butter weakens the peroxidases, but has little 
effect on the catalase and galactase. 

(7) Bnzyms are present in butter in such small amounts and under 
such unfavorable conditions for enzym activity during cold storage 
that they need not be considered as a factor in the deterioration of 
butter during storage. 

LITERATURE CITED 

(1) Babcock, S. M., and Russell, H. L. 

1897. UNORGANIZED FERMENTS OP MILK: A NEW Fx\CTOR IN THE RIPENING OF 

cheese. In Wis. Agr. Exp. Sta., 14th Ann. Rpt., [i896]/97, p. 161-193, 
fig. 14-15. 
(2) and Vivian, Alfred. 

1898. distribution of galactase in cow's milk. In Wis. Agr. Exp. Sta., 

15th Ann. Rpt., [18971/98, p. 87-92, fig. 13. 

(3) * 

1898. properties oe galactase: a digestive ferment of milk. In Wis. 

Agr. Exp. Sta., 15th Ann. Rpt., [18971/98, p. 77-86. 
(4) 

1899. influence of galactase in the ripening of cottage cheese. In Wis. 

Agr. Exp. Sta., 16th Ann. Rpt., [18981/99, p. 175-178. 

(5) and others. 

1899. THE ACTION OF PROTEOLYTIC FERMENTS ON MILK, WITH SPECIAL REFERENCE 
TO GALACTASE, THE CHEESE-RIPENING ENZYME- In Wis. Agr. Exp . 

Sta., 16th Ann. Rpt., [18981/99, p. 157-174, fig. 31-42. 

(6) BarthEL, Christian. 

I91O. METHODS USED IN THE EXAMINATION OF MILK AND DAIRY PRODUCTS. 

Translated by W. Goodwin. 260 p., 65 fig. London. 

(7) Bunzel, H. H. 

1914. A SIMPLIFIED AND INEXPENSIVE OXIDASE APPARATUS. In Jour. Biol. 

Chem., v. 17, no. 3, p. 409-411, 1 fig. 

(8) EulER, Hans. 

1912. general chemistry of the Enzymes. Translated by T. H. Pope. 
323 p., 7 fig. New York, I^ondon. 



Nov. 2 6, 1917 Enzyms of Milk and Butter 449 

(9) GillET, Charles. 

1904. THE PRESENCE OF a lipase in milk. (Abstract.) In Exp. Sta. Rec, 
v. 15, no. 10, p. 1002. (Original article in Jour. Physiol, et Pathol. 
Gen., 1903, no. 3. Not seen.) 

(10) Grimmer, W. 

1910. the Enzyms OP THE mammary glands. (Abstract.) In Exp. Sta. 

Rec, v. 23, no. 3, p. 285-286. (Original article in Festschrift Otto 
Wallach, p. 452-466. Gottingen, 1909. Not seen.) 

(11) Harding, V. J., and Maclean, R. M. 

1915. A COLORIMETRIC METHOD POR THE ESTIMATION OF AMINO-ACID a-NITRO- 

GEN. In Jour. Biol. Chem., v. 20, no. 3, p. 217-230, 3 fig. 

(12) Hesse. 

1913. caTalasE IN butter. (Abstract.) In Exp. Sta. Rec, v. 29, no. 6, 
p. 508. (Original article in Molk. Ztg. [Hildesheim], Bd. 26, No. 6, 
p. 81-84, 1912. Not seen.) 

(13) KoopER, W. D. 

1912. investigations in regard To caTalasE. (Abstract.) In Exp. Sta. 

Rec, v. 26, no. 2, p. 112. (Original article in Milchw. Zentbl., Bd* 
7, No. 6, p. 264-271, 1 fig., 1911. Not seen.) 

(14) Olson, G. A. 

1908. milk proteins. In Jour. Biol. Chem., v. 5, No. 2/3, p. 261-281. 

(15) Rahn, Otto, Brown, C. W., and Smith, I,. M. 

1909. keeping qualities of butter. Mich. Agr. Exp. Sta. Tech. Bui. 2, 

44 p., 6 fig. Literature cited, p. 44. 

(16) Rogers, L. A. 

1904. STUDIES UPON THE KEEPING QUALITY OF BUTTER. I. CANNED BUTTER. 

U. S. Dept. Agr. Bur. Anim. Indus. Bui. 57, 24 p. Bibliography, 
p. 24. 

(17) and others. 

19 13. FACTORS INFLUENCING THE CHANGE IN FLAVOR IN STORAGE BUTTER. 

U. S. Dept. Agr. Bur. Anim. Indus. Bui. 162, 69 p., 1 fig. 

(18) SpindlER, Franz. 

191 1. beitrage zur kenntnis der milchkatalase. In Biochem. Ztschr., 

Bd. 30, Heft 5, p. 384-412, 1 fig. 

(19) Spolverini, L. M. 

1904. THE oxidizing ferment in milk. (Abstract.) In Exp. Sta. Rec, v. 15, 

no. 10, p. 1002. (Original article in Rev. Hyg. et Med. Infantiles, t. 3, 
no. 2, p. 113-155, 1904. Not seen.) 

(20) STETTER, A. 

191 5. CATALASE AND REDUCTASE DETERMINATION IN COW'S MILK IN PRACTISE, 
AND THE RELATION BETWEEN CATALASE AND REDUCTASE ON THE ONE 
HAND AND THE SPECIFIC GRAVITY, FAT, AND ACIDITY ON THE OTHER. 

(Abstract.) In Exp. Sta. Rec, v. 33, no. 5, p. 414. (Original article 
in Milchw. Zentbl., Bd. 43, No. 14, p. 369-381, 2 fig., 1914. Not seen.) 

(21) Stoklasa, J., and others. 

1905. ON THE ISOLATION OF FERMENTATIVE ENZYMS FROM COWS* MILK. (Ab- 

stract.) In Exp. Sta. Rec, v. 16, no. 7, p. 700-701. (Original article 
in Ztschr. Landw. Versuchw. Oesterr., Bd. 7, No. 11, p. 755-774, 1 fig., 
1904.) 

(22) VandEvelde, A. J. J. 

1907. NOUVELLES RECHERCHES SUR LES FERMENTS SOLUBLES DE LAIT. 85 p. 

Bruxelles. 



450 Journal of Agricultural Research vol. xi, No. 9 

(23) Van de Velde, H., and LandsheerE, J. de. 

1904. THE FERMENTS OF MILK: AN EXPERIMENTAL AND CRITICAL STUDY. (Ab- 

stract.) In Exp. Sta. Rec, v. 15, no. 10, p. 1003. (Original article in 
Ann. Soc. Medico-Chirurg. Anvers, 1903. Not seen.) 

(24) Van Slyke, D. D. 

1910. eine methode zur quantitativen bestimmung der aliphatischen 
aminogruppen ; einige anwendungen derselben in der chemie 
DER PROTEiNE DES harns und DER enzyme. In Ber. Deut. Chem. 
Gesell., Jahrg. 43, No. 16, p. 3170-3187, 1 fig. 

(25) Washburn, R. M., and Dahlberg, A. C. 

191 7. the influence of salt on the changes taking place in storage 
butter. In Jour. Dairy Sci., v. 2, no. 2, p. 114-126. References, 
p. 126. 

(26) Wiley, H. W., ed. 

1908. OFFICIAL AND PROVISIONAL METHODS OF ANALYSIS, ASSOCIATION OF 
OFFICIAL AGRICULTURAL CHEMISTS. AS COMPILED BY THE COMMITTEE 

on revision OF methods. U. S. Dept. Agr. Bur. Chem. Bui. 107 
(rev.), 272 p., 13 fig. Reprinted in 1912. 
^27) ZaitschEk, A., and Szontagh, F. von. 

1905. COMPARATIVE INVESTIGATIONS ON THE CONTENT OF PROTEOLYTIC AND 

AMYLOLYTIC ENZYMS IN DIFFERENT KINDS OF MILK. (Abstract.) In 

Exp. Sta. Rec, v. 16, no. 6, p. 597. (Original article in Arch. Physiol. 
[Pfiuger], Bd. 104, No. 9/12, p. 539-549, 1904. Not seen.) 




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